Use of cyclopeta[g]quinazoline derivatives for treating cancer

ABSTRACT

Cyclopenta[g]quinazolines of the formula (I): wherein; A is hydrogen or a group OR or NR 0 R 1  wherein R 0  and R 1  are each independently hydrogen C 1-4  alkyl, C 1-4  alkenyl, C 1-4  alkynyl, C 2-4  hydroxyalkyl, C 1-4  halogenoalkyl or C 1-4  cyanoalkyl, or R 0  and R 1  together with the intermediate N form a five- or six-membered heterocyclic ring, p is an integer in the range  1  to  4;  R 2  is hydrogen, C 1-4  alkyl, C 3-4  alkenyl, C 3-4  alkynyl, C 2-4  hydroxyalkyl, C 2-4  halogenoalkyl or C 1-4  cyanoalkyl; Ar 1  is phenylene, thiophenediyl, thiazolediyl, pyridinediyl or pyrimidinediy which may optionally bear one or two subtituents selected from halogeno, hydroxy, amino, nitro, cyano, trifluoromethyl, C 1-4  alkyl and C 1-4  alkoxy; and R 3  is a group the following formula: (II) and pharmaceutically acceptable salts or calers may be used in the treatment of solid tumours.

This invention relates to the use of compounds as anti-cancer agents.More particularly it relates to cyclopenta[g]quinazoline derivativeswhich possess antiproliferative activity in the treatment of solidtumours.

One group of anti-cancer agents comprises antimetabolites havingantifolate activity, such as the dihydrofolate reductase inhibitor,methotrexate and the thymidylate synthase (TS) inhibitors CB3717,raltitrexed and ZD9331. CB3717 is described and claimed in EP-B-0031237,raltitrexed in EP-B-0239362 and ZD9331 in EP-B-0562734. All of these TSinhibitors have demonstrable clinical activity in a range of solidtumours (see Cancer Treatment Reports, 1986, 70, 1335 and Beale et al.,“Tomudex: Clinical Development” in Antifolate Drugs in Cancer Therapy(ed. Jackman), Humana Press, Totowa, N.J., USA, pp. 177-181, 1999).Side-effects of raltitrexed and ZD9331 are predominantly related toinhibition of TS in gut and bone-marrow.

TS catalyses the methylation of deoxyuridine monophosphate to producethymidine monophosphate which is required for DNA synthesis. Theanticancer activity of these agents may be assessed in vitro bydetermining their inhibitory effect on that enzyme, and in cell culturesby their inhibitory effect on a range of mouse and human cancer celllines (see Boyle et al, “ZD9331: Preclinical and clinical studies” inAntifolate Drugs in Cancer Therapy (cd. Jackman), Humana Press, Totowa,N.J., USA, pp. 243-260, 1999 and Hughes et al., “Raltitrexed (Tomudex),a highly polyglutamatable antifolate thymidylate synthase inhibitor:design and preclinical activity” in Antifolate Drugs in Cancer Therapy(ed. Jackman), Humana Press, Totowa, N.J., USA, pp. 147-165, 1999).

More recently, cyclopenta[g]quinazoline derivatives showing a good levelof activity both as regards their ability to inhibit TS and also asregards their anticancer activity against various cell lines have beendeveloped.

WO-A-94/11354 (British Technology Group Limited) discloses tricycliccompound of formula:

wherein R¹ is hydrogen, amino, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄hydroxyalkyl or C₁₋₄ fluoroalkyl;

-   -   R² is hydrogen, C₁₋₄ alkyl, C₃₋₄ alkenyl, C₃₋₄ alkynyl, C₂₋₄ A        hydroxyalkyl C₂₋₄ halogenoalkyl or C₁₋₄ cyanoalkyl;    -   Ar is phenylene, thiophenediyl, thiazolediyl, pyridinediyl or        pyrimidinediyl which may optionally bear one or two substituents        selected from halogeno, hydroxy, amino, nitro, cyano,        trifluoromethyl, C₁₋₄ alkyl and C₁₋₄ alkoxy; and    -   R³ is a group of one of the following formulae:        —NHCH(CO₂H)—A¹ —Y¹—NH—A³—Y³    -   or R³ is a N-linked naturally-occurring amino acid selected from        the group consisting of L-alanine, L-leucine, L-isoleucine,        L-valine and L-phenylalanine. Among the compounds disclosed is        the L-Glu-γ-D-Glu compound CB300638, also mentioned in Clinical        Cancer Research, 5, November 1999 (Supplement) at #566 (Theti et        al.) and Proceedings of the American Association for Cancer        Research, 41, March 2000 at #33 (Jackman et al.), as well as        in J. Med. Chem., 2000, 43, 1910-1926, where it is disclosed on        page 1923 as compound 7b.

WO-A-95/30673 (British Technology Group Limited) disclosescyclopenta-[g]quinazolines of formula:

wherein R¹ is hydrogen, amino, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄hydroxyalkyl or C₁₋₄ fluoroalkyl;

-   -   R² is hydrogen, C₁₋₄ alkyl, C₃₋₄ alkenyl, C₃₋₄ alkynyl, C₂₋₄        hydroxyalkyl, C₂₋₄ halogenoalkyl or C₁₋₄ cyanoalkyl;    -   Ar¹ is phenylene, thiophenediyl, thiazolediyl, pyridinediyl or        pyrimidinediyl which may optionally bear one or two substituents        selected from halogeno, hydroxy, amino, nitro, cyano,        trifluoromethyl, C₁₋₄ alkyl and C₁₋₄ alkoxy; and    -   R³ is a group of one of the following formulae:        —A¹—Ar²—A²—Y¹ —A—CON(R)CH(Y⁴)Y⁵ —A⁸—X—Ar⁴

The α-isoform of the folate receptor (α-FR; membrane-associatedfolate-binding protein) is a glycosylphosphatidylinositol anchored cellmembrane protein that has very high affinity for folic acid and the morebiologically relevant reduced-folates (Kd˜0.1 nM). The mechanism offolate internalisation is receptor-mediated endocytosis. The α-FR isoverexpressed in many carcinomas, particularly those of ovarian originwhere it is overexpressed highly and homogeneously in 90% of cases; seeCancer Res. 51, 5329-5338, 1991 (Campbell et al, 1991). Furthermore,high α-FR expression has been linked to aggressive, platinum resistantdisease and poor prognosis—see Int. J. Cancer 74, 193-198, 1997 and Int.J. Cancer 79, 121-126, 1998 both Toffoli et al.). The β-isoform iswidely expressed in tumours of epithelial and non-epithelial origin withexpression levels being generally low/moderate and high, respectively,reviewed in Critical Rev. Therap. in Drug Carrier Systems 15, 587-627,1998 (Reddy and Low).

Polate receptors (α and β) are expressed in some adult normal tissues(low to moderate expression). Significant expression of the α-FR islargely restricted to kidney proximal tubules and choroid plexusalthough it is suggested that it is localised to the apical membranesurface in these organs and therefore may not play a significant role infolate uptake from blood (Reddy and Low, ibid.). There may be aspecialised function of the α-FR in the proximal tubules of the kidneyto salvage folates that escape in the filtrate.

The α-FR is hypothesised to be involved in cell signalling pathways. Forexample, in IGROV-1 ovarian carcinoma cells, immunoprecipitationexperiments have shown that the α-FR is associated in membranes with theG protein G_(α1-3), and the non-receptor kinase lyn.

High FR expression in some tumours relative to normal tissues is beingexploited in several areas of cancer medicine, including the selectivetumour delivery of conjugates of folic acid and toxins, liposomes,imaging or cytotoxic agents (Reddy and Low, ibid.). For example, folicacid-deferroxamine-^(III)In conjugates are detected only inFR-expressing tumours and not normal tissues of mice, with the exceptionof kidney epithelial cells. The high selectivity of this approachresides in the very low and high affinities of folic acid (not a majorcomponent of plasma) for the RFC (reduced-folate carrier) and FRrespectively. Thus antifolate drugs with similarly low and high affinityfor the RFC and α-FR respectively could be highly selective for α-FRover-expressing tumours relative to normal tissues. In contrast with thefolic acid conjugates they would not require intracellular cleavage tobe active.

We have now discovered that certain compounds within the general classof cyclopenta[g]quinazolines have an unexpectedly high level ofselectivity for α-folate receptor expressing human tumour cell lines.Accordingly the present invention comprises the use of acyclopenta[g]quinazoline of formula (I):

wherein:

-   -   A is hydrogen or a group OR⁰ or NR⁰R¹ wherein R⁰ and R¹ are each        independently hydrogen, C₁₋₄ alkyl, C₃₋₄ alkenyl, C₃₋₄ alkynyl,        C₂₋₄ hydroxyalkyl, C₂₋₄ halogenoalkyl or C₁₋₄ cyanoalkyl, or R⁰        and R¹ together with the intermediate N form a five- or        six-membered heterocyclic ring;    -   p is an integer in the range 1 to 4;    -   R² is hydrogen, C₁₋₄ alkyl, C₃₋₄ alkenyl, C₃₋₄ alkynyl, C₂₋₄        hydroxyalkyl, C₂₋₄ halogenoalkyl or C₁₋₄ cyanoalkyl;    -   Ar¹ is phenylene, thiophenediyl, thiazolediyl, pyridinediyl or        pyrimidinediyl which may optionally bear one or two substituents        selected from halogeno, hydroxy, amino, nitro, cyano,        trifluoromethyl, C₁₋₄ alkyl and C₁₋₄ alkoxy; and    -   R³ is a group of the formula:        —A⁵—CON(R)CH(Y⁴)Y⁵    -   in which A⁵ is a C₁₋₆ alkylene group and R is hydrogen, C₁₋₄        alkyl, C₃₋₄ alkenyl or C₃₋₄ alkynyl;    -   Y⁴ is carboxy, tetrazol-5-yl, N-(C₁₋₄ alkylsulfonyl)carbamoyl,        N-(phenylsulfonyl)carbamoyl which may optionally bear one or two        substituents on the phenyl ring selected from the group        consisting of halogeno, nitro, C₁₋₄ alkyl and C₁₋₄ alkoxy,        tetrazol-5-ylthio, tetrazol-5-ylsulfinyl or        tetrazol-5-ylsulfonyl; and    -   Y⁵ is the residue of a naturally occurring amino acid        NH₂CH(CO₂H)Y⁵; or    -   Y⁵ is a group of the formula:        —A⁴—CO₂H    -   in which A⁴ is a C₂₋₆ alkylene group; or    -   Y⁵ is a group of the formula:        —A⁵—Ar³—A⁷—Y⁶    -   in which A⁶ is a bond between the α-carbon atom of the group        —A⁵—CON(R)CH(Y⁴)—and Ar³ or is a C₁₋₂ alkylene group;    -   Ar³ is phenylene, tetrazolediyl, thiophenedlyl, thiazolediyl,        pyridinediyl or pyrimidinediyl which in the case of phenylene        may optionally bear one or two substituents on the ring selected        from halogeno, nitro, C₁₋₄ alkyl and C₁₋₄ alkoxy,    -   A⁷ is a C₁₋₃ alkylene or C₂₋₃ alkenylene group; and    -   Y⁶ is carboxy, tetrazol-5-yl, N-(C₁₋₄ alkylsulfonyl)carbamoyl,        N-(phenylsulfonyl)carbamoyl which may optionally bear one or two        substituents on the phenyl ring selected from the group        consisting of halogeno, nitro, C₁₋₄ alkyl and C₁₋₄ alkoxy,        tetrazol-5-ylthio, tetrazol-5-ylsulfinyl or        tetrazol-5-ylsulfonyl;    -   the compound (I) optionally being in the form of a        pharmaceutically acceptable salt or ester;    -   for the manufacture of a medicament for the treatment of solid        tumours.

The compounds of the invention display one or more of the followingadvantages:

-   -   1. They have high selectivity for tumours over-expressing the        α-FR, when grown in physiological concentrations of folate and        possessing normal expression of the RFC.    -   2. They display a potent TS inhibition, a low affinity for the        RFC and a moderate to high affinity for the α-FR (inverse        relative affinity of approx. 0.1 to 1.5).    -   3. They have TS-specific activity and are resistant to in vivo        hydrolases.    -   4. They display selective activity (in the order of >100-fold)        in primary human cell line screen with high α-FR expression        (A431/A431-FBP isogenic pair and KB cells=folic acid). They        display selective activity (in the order of >10-fold) in cell        lines with moderate FR expression. They display tumour-specific        localisation in mice. They display antitumour activity in        xenografts with no toxicity.

In this specification the terms alkyl, alkenyl, alkynyl and alkyleneinclude both straight and branched chain groups but references toindividual alkyl or alkylene groups, such as “propyl”, are specific forthe straight chain group only. An analogous convention applies to othergeneric terns. Moreover, the numbering system used for thecyclopenta[g]quinazoline nucleus is the conventional one as shown below:

Amino-acid residues are designated herein in the standard manner (Pureand Applied Chemistry, 1974, 40, 317 and European Journal ofBiochemistry, 1984, 138, 9). Thus, for example, γ-glutamyl denotes theradical H₂NCH(CO₂H)CH₂CH₂CO— or —NHCH(CO₂H)CH₂CH₂CO— according to thecontext, the carbon atoms in these radicals being numbered from thecarbon atom of the α-carboxy group as position 1.

It will be observed that a cyclopenta[g]quinazoline of the inventioncontains at least two asymmetric carbon atoms [present at the point ofattachment of the group —N(R²)— to the tricyclic ring system and at theα-carbon atom of the group —CONHCH(CO₂H)—] and can therefore exist inracemic and optically active forms. It is to be understood that thisinvention encompasses both racemic and optically active forms of thephysiologically active cyclopenta[g]quinazolines, it being a matter ofcommon general knowledge how such optically active forms may be obtainedby stereospecific synthesis or by separation of a mixture of isomericcompounds. It will be appreciated that one isomer may be of moreinterest than another due to the nature of the activity which itexhibits or due to superior physical properties, for example aqueoussolubility.

It is also to be understood that a cyclopenta[g]quinazoline of theformula (I) may exhibit the phenomenon of tautomerism and that theformulae shown in this specification represent only one of the possibletautomeric forms. Moreover, it will be appreciated that when, forexample, Y₄ or Y₆ is a tetrazol-5-yl group, that group may be in theform of a 1H-tetrazol-5-yl group or a 2H-tetrazol-5-yl group. It is tobe understood therefore that the invention is not limited merely to anyone tautomeric form which is illustrated.

It is also to be understood that certain cyclopenta[g]quinazolines ofthe formula (I) can exist in solvated as well as unsolvated forms suchas, for example, hydrated forms.

A suitable value for R⁰, R¹ or R² when it is C₁₋₄alkyl, or for a C₁₋₄alkyl substituent which may be present on Ar¹ or Ar₃ or on a phenylgroup-containing group Y⁴ or Y⁶ present in R³, or for a group R presentin R³ when it is C₁₋₄ alkyl, is, for example, methyl, ethyl, propyl orisopropyl.

A suitable value for a C₁₋₄ alkoxy substituent which may be present onAr¹ or Ar³ or on a phenyl-containing group Y⁴ or Y⁶ is, for example,methoxy, ethoxy, propoxy, isopropoxy or butoxy.

A suitable value for a halogeno substituent which may be present on Ar¹or Ar³ or on a phenyl-containing group Y⁴ or Y⁶ is, for example, fluoro,chloro or bromo.

A suitable value for R⁰, R¹ and R² when it is C₃₋₄ alkenyl or for agroup R present in R³ when it is alkenyl, is, for example, prop-2-enyl,but-2-enyl, but-3-enyl or 2-methylprop-2-enyl; and when it is C₃₋₄alkynyl is, for example, prop-2-ynyl or but-3-ynyl.

A suitable value for R⁰, R¹ and R² when it is C₂₋₄ hydroxyalkyl is, forexample, 2-hydroxyethyl or 3-hydroxypropyl; when it is C₂₋₄halogenoalkyl is, for example, 2-fluoroethyl, 2-chloroethyl,2-bromoethyl, 3-fluoropropyl, 3-chloropropyl or 3-bromopropyl; and whenit is C¹⁻⁴ cyanoalkyl is, for example, cyanomethyl, 2-cyanoethyl or3-cyanopropyl.

When R⁰ and R¹ together with the intermediate N form a five- orsix-membered heterocyclic ring, this may bear substituents, but the ringis preferably an unsubstituted saturated ring such as pyrrolidine orpiperidine.

A suitable value for Ar¹ or Ar³ when it is phenylene is, for example,1,3- or 1,4phenylene, especially 1,4-phenylene.

A suitable value for Ar¹ or Ar³ when it is thiophenediyl is, forexample, thiophene-2,4-diyl or thiophene-2,5-diyl; when it isthiazolediyl is, for example thiazole-2,4-diyl or thiazole-2,5-diyl;when it is pyridinediyl is, for example, pyridine-2,4-diyl,pyridine2,5-diyl, pyridine-2,6diyl or pyridine-3,5-diyl; and when it ispyrimidinediyl is, for example, pyrimidine-2,4-diyl, pyrimidine2,5-diylor pyrimidine-4,6-diyl.

As indicated, Ar¹ and a phenylene group Ar³ may carry one or twosubstituents. A preferred level of substitution in Ar¹, wheresubstitution is present, is either two substituents or especially onesubstituent; and the one or two substituents may conveniently be atpositions adjacent to the atom bonded to the group —CONHCH(CO₂H)—R³,halogeno substituents such as fluoro being preferred. A preferred levelof substitution on a phenylene group Ar^(3,) where substitution ispresent, is one substituent.

A preferred value for Y⁴ or y⁶ is tetrazol-5-yl or especially carboxy.

A suitable value for A⁵ is, for example, methylene, ethylene,trimethylene, tetramethylene, pentamethylene or hexamethylene and asuitable value for R is as described hereinbefore. A suitable value forY⁴ when it is N-(C₁₋₄ alkylsulfonyl)-carbamoyl is, for example,N-methylsulfonylcarbamoyl, N-ethylsulfonylcarbamoyl orN-propylsulfonylcarbamoyl.

A suitable value for Y⁵ when it is the residue of a naturally occurringamino acid is alanine (Y₅=CH₃), arginine (Y⁵=(CH₂)₃NHC(NH₂)=NH),aspartic acid (Y⁵=CH₂CO₂H), cysteine (Y⁵=CH₂SH), isoleucine(Y⁵=CH(CH₃)CH₂CH₃), leucine (y⁵=CH₂CH(CH₃)CH₃), omithine Y⁵=(CH₂)₃NH₂),phenylalanine (Y⁵=CH₂C₆H₅), serine (Y⁵=CH₂OH), valine (Y⁵=CH(CH₃)₂), andespecially glutamic acid (Y⁵=CH₂CH₂CO₂H). When Y⁵ is a group of theformula —A⁴CO₂—H, a suitable value for A⁴ is trimethylene,pentamethylene or bexamethylene, A⁴ preferably being a C₃₋₆ alkylenegroup with especially suitable values for Y⁵ being —(CH₂)_(n)CO₂H wheren is 3,4 or 5.

A suitable value for A⁶ in a group Y⁵ of the formula —A⁶Ar³—A⁷—Y⁶ whenit is a C₁₋₂ alkylene group is, for example methylene or ethylene, andfor A⁷ when it is a C₁₋₃ alkylene group is, for example, methylene,ethylene or trimethylene. A suitable value for A⁷ when it is a C₂₋₃alkenylene group is, for example, vinylene or especially propenylene(—CH₂CH═CH— or —CH═CH=CH₂). Suitable values for Ar₃ include those whichhave been discussed hereinbefore, such as thiophenediyl or mostespecially phenylene, or additionally tetrazole- 1,5-diyl ortetrazole-2,5-diyl. A suitable value for Y⁶ in such a group Y⁵ when itis N—(C₁₋₄ alkylsulfonyl)carbamoyl is, for example,N-methylsulfonylcarbamoyl, N-ethylsulfonylcarbamoyl orN-propylsulfonylcarbamoyl.

A suitable pharmaceutically-acceptable salt form of acyclopenta[g]-quinazoline of the invention is, for example, an acidaddition salt with an inorganic or organic acid, for examplehydrochloric, hydrobromic, trifluoroacetic or maleic acid; or an alkalimetal, for example sodium, an alkaline earth metal, for example calcium,or ammonium, for example tetra(2-hydroxyethyl)ammonium, salt.

A suitable pharmaceutically-acceptable ester form of acyclopenta[g]-quinazoline of the invention is, for example, an esterwith an aliphatic alcohol of up to 6 carbon atoms, for example a methyl,ethyl or tert-butyl ester.

It is to be understood that R³ may contain several carboxy groups inaddition to the carboxy group in the grouping —ONHCH(CO₂H)— When, forexample, two carboxy groups are present in the cyclopenta[g]quinazoline,a salt or ester may be mono-acid-mono-salt or -ester, di-salt ordi-ester and when, for example, three carboxy groups are present a saltor ester may be mono-acid-di-salt or -ester, di-acid-mono-salt or -esteror even tri-salt or -ester.

Particularly preferred values for the various symbols R⁰, R¹, R² and Ar¹individually are as expressed for the preferredcyclopenta[g]quinazolines described hereinafter.

A preferred cyclopenta[g]quinazoline of the invention has the formula(I) wherein R⁰ and R¹ are each independently hydrogen or C₁₋₄ alkyl,especially methyl;

-   -   R² is ethyl, propyl, prop-2-enyl, prop-2-ynyl, 2-hydroxyethyl,        2-fluoroethyl, 2-bromoethyl or 2-cyanoethyl;    -   Ar¹ is 1,4-phenylene which may optionally bear one or two        substituents selected from the group consisting of chloro and        especially fluoro, thiophene-2,5-diyl, thiazole-2,5-diyl or        pyridine-2,5-diyl;    -   A⁵ is an ethylene group; and    -   Y⁵ is the residue of a naturally occurring amino acid        NH₂CH(CO₂H)Y⁵.    -   A preferred value for p is 1.

A further preferred cyclopenta[g]quinazoline of the invention has theformula (I) wherein A is hydrogen or a group OR⁰ in which R⁰ is hydrogenor methyl;

-   -   R² is ethyl or prop-2-ynyl;    -   Ar¹ is 1,4-phenylene or 1,4-phenylene having a 2-fluoro        substituent as in 2,6-difluoro-1,4-phenylene or especially        2-fluoro-1,4-phenylene or is pyridine 2,5-diyl; and    -   A⁵ and Y⁵ are just as described above.

An especially preferred cyclopenta[g]quinazoline of the invention hasthe formula (I) wherein A is hydrogen or a group OR⁰ in which R⁰ ishydrogen or methyl;

-   -   wherein R² is ethyl or preferably prop-2-ynyl;    -   Ar¹ is 1,4-phenylene or 2-fluoro- 1,phenylene; and    -   R³ is the residue of an acid NH₂CH(COOH)R³ which comprises        L-Glu-γ-D-Glu, i.e. N-L-γ-glutamyl-D-glutamic acid, or        L-Glu-γ-L-Glu, i.e. N-L-γ-glutamyl-L-glutamic acid wherein R³ is        of formula:    -   R³ is the corresponding N-methyl derivative of formula:    -   or R³ is the corresponding tetrazol-5-yl derivative of formula:

Other quinazolines of the invention of particular interest have thevalues of R⁰, R¹, R², and Ar¹ and Ar in combination as indicated abovebut with R³ having any value as indicated hereinbefore. However,specific particularly preferred cyclopenta-[g]quinazolines of theinvention are:

-   -   N-{-{4-[N-((6RS)-2-methyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}-D-glutamic        acid;    -   N-{N-{2-fluoro-4-[N-((6RS)-2-methyl4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}-D-glutamic        acid;    -   (4R)-4-{N{N-{4-[N-((6RS)-2-methyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}-amino}-4-(tetrazol-5-yl)butyric        acid;    -   N-{N-{4-[N-((6RS)-2-hydroxymethyl4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}-D-glutamic        acid;    -   N-methyl-N-{N-{4-[N-((6RS)-2-methyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}-L-glutamic        acid; and    -   N-{N-{4-[N-((6RS)-2-hydroxymethyl-4oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}-N-methyl-L-glutamic        acid;    -   or a pharmaceutically acceptable salt or ester thereof.

Although the compounds of the present invention can exist as a mixtureof stereoisomers it is preferred that they are resolved into oneoptically active isomeric form. Such a requirement complicates thesynthesis of the compounds and it is preferred therefore that theycontain as few asymmetric carbon atoms as possible consistent withachieving the desired activity.

As indicated previously, however, the cyclopenta[g]quinazolines of thepresent invention contain at least two asymmetric carbon atoms. Ofthese, that at the 6 position of the ring system preferably has the 6Sorientation rather than the 6R orientation, whilst the alpha carbon atomof the group —CONHCH(CO₂H)— preferably has the L rather than the Dconfiguration. The preferred compounds (I) described hereinbefore thuspreferably have such a configuration at these two asymmetric carbonatoms or less preferably are a racemic mixture in which one or both ofthese asymmetric carbon atoms is unresolved.

The asymmetric carbon atom of a residue —A⁵—CON(R)CH(Y⁴)Y⁵ may be of theL- or D-configuration but the amide bond will be stabilised in vivo whenit is of the D-configuration as it will also be when R is other thanhydrogen. When Y⁵ is the residue of a naturally occurring amino acid,however, the amino acid intermediate for the synthesis of thecyclopenta[g]quinazoline will of course be more readily available whenthis asymmetric carbon atom is of the L-configuration.

A cyclopenta[g]quinazoline of the invention may be prepared by anyprocess known to be applicable to the preparation of chemically-relatedcompounds.

The anti-cancer activity of the cyclopenta[g]quinazolines of the presentinvention may be assessed, for example, using one or more of theprocedures set out below:

-   -   (a) An in vitro assay which determines the ability of a test        compound to inhibit the enzyme thymidylate synthase. Thymidylate        synthase may be obtained in partially purified form from L1210        mouse leukaemia cells and utilised in the assay using the        procedures described by Jackman et al. (Cancer Res., 1986,        46, 2810) and Sikora et al. (Biochem. Pharmacol., 1988, 37,        4047),    -   (b) An assay which determines the ability of a test compound to        bind to the α-FR relative to that of folic acid, using mouse        L1210FBP cells (α-FR expression) in the procedure described by        Westerhof et al. (Cancer Res., 1991, 51, 5507-5513);    -   (c) An assay which determines the ability of a test compound to        inhibit the growth of human tumour cell lines expressing the        α-FR (A431-FBP vulvular carcinoma transfected with the α-FR; KB        nasopharengeal carcinoma);    -   (d) An assay which determines the ability of a test compound to        inhibit the growth of human tumour cell lines not expressing the        α-FR (A431 neo-transfected);    -   (e) An assay confirming or demonstrating that compound-induced        growth inhibition is largely attributable to α-FR mediated        uptake into KB or A431-FBP cells. This involves the co-addition        of an excess of folic acid (1 μM) to compete with the compounds        for FR but not RFC binding.

Although the pharmacological properties of the cyclopenta[g]quinazolinesof the invention depend on their detailed structure, in general thecyclopenta[g]quinazolines of the invention possess activity in one ormore of the above tests (a) to (d) as indicated below:

-   -   Test (a) IC₅₀ in the range, for example, 0.0001-1 μM;    -   Test (b) Inverse relative affinity in the range, for example,        0.05-5 (values greater than 1 implies binding is greater than        that of folic acid and values less that 1 implies binding weaker        than that of folic acid);    -   Test (c) IC₅₀ in the range, for example, 0.001-10 μM;    -   Test (d) IC₅₀ in the range, for example, 0.01-100 μM;    -   Test (e) IC₅₀ at least 10-fold higher than that seen in test (b)        for the same cell line.

A cyclopenta[g]quinazoline of the present invention may itself be activeor it may be a pro-drug which is converted in vivo to an activecompound. A cyclopenta[g]quinazoline of the invention may beadministered to a warm-blooded animal, including a human, in the form ofa pharmaceutical composition which comprises thecyclopenta[g]quinazoline in association with apharmaceutically-acceptable diluent or carrier.

The composition may be in a form suitable for oral use, for example atablet, capsule, aqueous or oily solution, suspension or emulsion; aform suitable for topical use, for example a cream, ointment, gel oraqueous or oily solution or suspension: a form suitable for nasal use,for example a snuff, nasal spray or nasal drops; a form suitable forvaginal or rectal use, for example a suppository, a form suitable foradministration by inhalation, for example as a finely divided powdersuch as a dry powder, a microcrystalline form or a liquid aerosol; aform suitable for sub-lingual or buccal use, for example a tablet orcapsule; or a form suitable for parenteral use (including intravenous,subcutaneous, intramuscular, intravascular or infusion use), for examplea sterile aqueous or oily solution, emulsion or suspension. In generalthe above compositions may be prepared in a conventional manner usingconvention excipients.

The composition may contain, in addition to the cyclopenta[g]quinazolineof the invention, one or more other anti-cancer substances selectedfrom, for example, other antimetabolites, DNA interacting agents, signaltransduction inhibitors or other inhibitors of deregulated pathways intumours.

The cyclopenta[g]quinazoline will normally be administered to awarm-blooded animal at a dose within a range of 50-25,000, particularly50-5,000, mg per square metre body area of the animal, i.e.approximately 1,500, particularly 1-100, mg/kg. Where desired, however,dosages outside this range may be employed and, in particular, where thepreferred mode of administration involving subcutaneous infusion is usedthen the does range may be increased to 1-1,000 mg/kg. Preferably adaily dose in the range 10-250 mg/kg is employed, particularly 30-150mg/kg. However, the daily dose will necessarily be varied depending uponthe host treated, the particular route of administration and theseverity of the illness being treated. Accordingly, the optimum dosagemay be determined by the practitioner who is treating any particularpatient.

Accordingly the present invention also includes a method for aidingregression and palliation of cancer in a patient, particularly awarm-blooded animal such as a human, in need of such treatment, whichcomprises administering to said patient an effective amount of acyclopenta[g]quinazoline as defined hereinbefore.

Cyclopenta[g]quinazolines of the present invention are of interest for awide range of anti-tumour activities against solid tumours, butparticularly the treatment of ovarian cancer.

In view of the activity shown by antimetabolites such as aminopterin andmethotrexate, which is discussed hereinbefore, thecyclopenta[g]quinazolines of the present invention are also of interestfor use in the treatment of other conditions, for example allergicconditions such as psoriasis and inflammatory diseases such asrheumatoid arthritis. In using a cyclopenta[g]quinazoline of theinvention for such a purpose the compound will normally be administeredat a dose within the range 5-25,000, particularly 5-500, mg per squaremetre body area of the animal, i.e. approximately 0.1-500, particularly0.1-10, mg/kg. Where desired, however, dosages outside this range may beemployed. In general, for the treatment of an allergic condition such aspsoriasis, topical administration of a cyclopenta[g]quinazoline of theinvention is preferred. Thus, for example, for topical administration adaily dose in the range, for example, of 0.1 to 10 mg/kg may be used.

Compositions containing the quinazolines may be formulated in unitdosage form, i.e. in the form of discrete portions each comprising aunit dose, or a multiple or sub-multiple of a unit dose, for example asa tablet or capsule. Such a unit dosage form may, for example, containan amount of the cyclopenta[g]quinazoline in the range of 1-250 or 1-500mg.

The invention is illustrated by the following Examples.

EXAMPLE 1 Synthesis of CB300945 (2-CH₂OH Derivative of CB300638)

2-Hydroxymethyl-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-4-one

A solution of caesium acetate (14.4 g, 75.2 mmol) in dry DMF (40 ml) washeated to 60° C. under argon for 30 min. The mixture was cooled to 40°C. and a suspension of2-chloromethyl-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-4one (L.Skelton, V. Bavetsias, A. Jackman, WO 00/050417-A1; 2.2 g, 9.4 mmol) indry DMF (60 ml) was added via a cannula. The mixture was heated to 80°C. under argon for 16 h. The mixture was cooled to room temperature andthe solvent was removed in vacuo. The residue was suspended in water (50ml) and MeOH (20 ml). The pH was adjusted to 12.5 with 1 M sodiumhydroxide solution and the brown suspension was stirred for 2 h at roomtemperature. The insoluble brown solid was removed by filtration and theresulting solution was acidified to pH 5 with 1 M hydrochloric acid. Theprecipitate was collected by filtration, washed with acidified water anddried in vacuo over P₂O₅ to yield the product as a pale yellow solid(1.17 g, 58%); m.p. 205-210° C.; ¹H NMR (DMSO-d₆) δ 2.07 (quin, J=7.4Hz, 2 H, 7-H), 2.98 (q, J=6.95 Hz, 4 H, 6-H and 8-H), 4.38 (s, 2H,2-CH₂), 7.46 (s, 1 H, 9-H), 7.92 (s, 1 H, 5-H); MS (FAB-m/z): Found 217[(M+H)⁺, 100%]; HRMS: measured 217.0977; calculated for C₁₂H₁₃N₂O₂(M+H)⁺: 217.0977; Found C, 64.01; H, 5.23; N, 12.34. C₁₂H₁₃N₂O_(2,)½H2Orequires C, 63.93; H, 5.77; N, 12.43%.

2-(2,2-Dimethylpropionyloxymethlyl)-3,4,7,8-tetrahydro-6H-cyclopenta-[g]quinazolin-4-one

2-Hydroxymethyl-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-4-one (1.0g, 4.6 mmol), triethylamine (0.77 ml, 5.6 mmol), DMAP (50 mg, 0.4 mmol)and anhydrous CH₂Cl₂ (50 ml) were mixed in a flask under argon. Pivalicanhydride (1.2 ml, 6.0 mmol) was added dropwise and the suspensionstirred at room temperature under argon for 5 h. The solvent was removedin vacuo and the residue partitioned between EtOAc (100 ml) andsaturated aqueous NaHCO₃ (100 ml). The organic extract was washed withsaturated aqueous NaHCO₃ (70 ml), water (70 ml), brine (70 ml), dried(Na₂SO₄) and the solvent removed in vacuo. The residue was trituratedwith hexane (60 ml) and the product collected by filtration as a yellowsolid (1.21 g, 87%); m.p. 185-190° C.; ¹H-NMR (DMSO-d₆) δ 1.22 (s, 9H,CMe₃), 2.07 (quin, J=7.4 Hz, 2 H, 7-H), 2.98 (q, J=5.72 Hz, 4 H, 6-H and8-H), 4.94 (s, 2 H, 2-CH₂), 7.42 (s, 1 H, 9-H), 7.92 (s, 1 H, 5-H),12.20 (br, 1 H, NH); MS (FAB, m/z): Found 301 [(M+H)⁺, 100%]; HRMS:measured 301.1539; calculated for C₁₇H₂₁N₂O₃ (M+H)⁺: 301.1552; Found C,67.65; H, 6.54, N, 9.54, C₁₇H₂₀N₂O₃ requires C, 67.98; H, 6.71; N,9.33%.

2-(2,2-Dimethylpropionyloxymethyl)-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-4,6-dioneand2-(2,2-Dimethylpropionyloxymethyl)-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-4,8dione

To a stirred solution of (Ph₃SiO)₂CrO₂ (L. M. Baker and W. L. Carrick,J. Org. Chem. 1970, 35, 774) (10.6 mg, 0.017 mmol) in CH₂Cl₂ (5 ml) wasadded sequentially aqueous 70% tert-butyl hydroperoxide (0.18 ml, 1.3mmol) and2-(2,2-dimethylpropionyloxymethyl)-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-4-one(0.1 g, 0.33 mmol). The mixture was stirred at room temperature withprotection from the light for 24 h. The solvents were removed in vacuoand the residue purified by column chromatography (20 g of silica gel)eluting with a gradient of 10-30% EtOAc in CHCl₃ to yield2-(2,2-dimethylpropionyloxymethyl)-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-4,6-dioneas a white solid (47 mg, 45%); m.p. 185-190C; ¹H-NMR (DMSO-d₆) δ 1.23(s, 9 H, CMe₃), 2.72 (m, 2 H, 7-H), 3.25 (m, 2 H, 8-H), 5.00 (s, 2 H,2-CH₂), 7.70 (s, 1 H, 9-H), 8.29 (s, 1 H, 5-H), 12.20 (br, 1 H, NH); MS(FAB, m/z): Found 315 [(M+H)⁺, 100%], 337 [(M+Na)⁺, 75%]; HRMS; measured315.1360; calculated for C₁₇H₁₉N₂O₄ (M+H)⁺: 315.1345; Found C, 64.18; H,5.72; N, 8.81. C₁₇H₁₈N₂O₄0.2H₂O requires C, 64.23; H, 5.79; N, 8.82%.

2-(2,2-Dimethylpropionyloxymethyl3,4,7,8-tetrahydro-6H-cyclopenta[g]-quinazolin-4,8-dione;¹H-NMR (DMSO-d₆) δ1.23 (s, 9H, CMe₃), 2.76 (m, 2 H, 7-H), 3.26 (m, 2 H,8-H), 4.98 (s, 2 H, 2-CH₂), 7.72 (s, 1 H, 9-H), 8.29 (s, 1 H, 5-H), 12.3(br, 1 H, NH).

Tert-Butyl 4-[N-((6RS)-2-(2,2-dimethylpropionyloxymethyl)-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)amino]benzoate

A suspension of2-(2,2dimethylpropionyloxymethyl)-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-4,6-dione(0.47 g, 1.50 mmol) in anhydrous methanol (33 ml) and anhydrous CH₂Cl₂(5 ml) was treated with tert-butyl 4-aminobenzoate (0.34 g, 1.78 mmol)followed by decaborane (0.07g, 0.58 mmol) and the mixture stirred atroom temperature under argon for 18 h. The solvent was removed in vacuoand the residue purified by column chromatography (50 g of silica gel)eluting with 30% ethyl acetate in CH₂Cl₂ to yield the desired product asa white solid (0.43 g, 58%); m.p. 231° C., ¹H-NMR (CDCl₃) δ1.26 (s, 9H,CMe₃), 1.58 (s, 9 H, CO₂CMe₃), 2.00 (m, 1 H, 7-H), 2.72 (m, 1 H, 7-H),3.08 (m, 2 H, 8-H), 5.10 (s, 2 H, 2-CH₂), 5.15 (m, 1 H, 6-H), 6.67 (d,J=8.8 Hz, 2 H, 3′-H, 5′-H), 7.58 (s, 1 H, 9-H), 7.87 (d, J=8.8 Hz, 2 H,2′-H, 6′-H), 8.24 (s, 1 H, 5-H); MS (FAB, m/z): Found 491 [(M+H)⁺, 25%],514 [(M+Na)⁺, 100%]; Found C, 68.37; H, 6.86; N, 8.35. C₂₈H₃₃N₃O₅requires C, 68.41; H, 6.77; N, 8.55%.

Tert-Butyl4-[N((6RS)-2-(2,2-dimethylpropionyloxymethyl)4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoate

A suspension of (propargyl)Co₂(CO)₆ ⁺BF₄ (213 mg, 0.52 mmol) inanhydrous CH₂Cl₂ (25 ml) was treated with tert-butyl4-[N-((6RS)-2-(2,2-dimethylpropionyloxymethyl)-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin6-yl)-amino]benzoate(200 mg, 0.41 mmol) and the red solution stirred at room temperatureunder argon for 15 minutes. Diisopropylethylamine (0.15 ml. 0.86 mmol)was added and the mixture stirred at room temperature under argon for 1h. The mixture was partitioned between ethyl acetate (30 ml) and brine(30 ml). The organic extract was dried (Na₂SO₄) and the solvent removedin vacuo. The residue was purified by column chromatography (20 g ofsilica gel) eluting with a gradient of 0-10% ethyl acetate in CH₂Cl₂ toyield the complex as a red oil (191 mg, 58%); ¹H-NMR (CDCl₃) δ 1.26 (s,9 H, CMe₃), 1.59 (s, 9 H, CO₂CMe₃), 2.31 (m, 1 H, 7-H), 2.62 (m, 1H,7-H), 3.13 (m, 2 H, 8-H), 4.57 (AB system, J=16.9 Hz, 2 H, propargylCH₂), 5,09 (s, 2 H, 2-CH₂), 5.63 (t, J=8.3, 1 H, 6-H), 5.98 (s, 1 H,propargyl CH), 6.91 (d, J=8. 9 Hz, 2 H, 3′-H, 5′-H), 7.61 (s, 1 H, 9-H),7.90 (d, J=8.9 Hz, 2 H, 2′-H, 6′-H), 8.14 (s, 1 H, 5-H), 10.25 (br s, 1H).

A solution of this complex (186 mg, 0.23 mmol) in ethanol (30 ml) wastreated with Fe(NO₃)₃.9H₂O (1.1 g) and the solution stirred at roomtemperature for 2 h. The solution was partitioned between ethyl acetate(30 ml) and water (30 ml). The organic extract was washed with brine (30ml), dried (Na₂SO₄) and the solvent removed in vacuo. The residue waspurified by column chromatography (20 g of silica gel) eluting with 10%ethyl acetate in CH₂Cl₂ to yield the desired product as a white solid(94 mg, 78%); m.p. 134° C.; ¹H-NMR (CDCl₃) δ 1.32 (s, 9 H, CMe₃), 1.61(s, 9H, CO₂CMe₃), 2.23 (s, 1H, propargyl CH), 2.38 (m, 1H, 7-H), 2.62(m, 1 H, 7-H), 3.07 (m, 1 H, 8-H), 3.25 (m, 1 H, 8-H), 3.94 (AB system,J=18.6 Hz, 2 H propargyl CH₂), 5.12 (s, 2 H, 2-CH2), 5.68 (t, J=8.2 Hz,1 H, 6-H), 6.99 (d, J=9.1 Hz, 2 H, 3′-H, 5′-H), 7.63 (s, 1 H, 9-H), 7.95(d, J=9.0 Hz) 2 H, 2′-H, 6′-H), 8.16 (s, 1 H, 5-H), 9.55 (br s, 1 H), MS(ESI, m/z) 552 {(M+Na)⁺, 100%}, 530 {(M+H⁺, 20%}; Found C, 70.14; H,6.80; N, 7.73. C₃₁H₃₅N₃O₅ requires C, 70.30; H, 6.66; N, 7.93%.

4-[N-((6RS)-2-(2,2-Dimethylpropionyloxymethyl)-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoicacid

A solution of tert-butyl4-[N-((6RS)-2-(2,2dimethylpropionyloxymethyl)-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop2-ynyl)amino]-benzoate(80 mg, 0.15 mmol) in trifluoroacetic acid (5 ml) was stirred at roomtemperature with protection from the light for 1.5 h. The solvent wasremoved in vacuo and the residue triturated with 1:1 diethyl ether andhexane to yield the desired product as a white solid (81 mg, TFA salt);m.p. 133° C., ¹H-NMR (DMSO-d6) δ 1.23 (s, 9H, CO₂CMc₃), 2.22 (m, 1 H,7-H), 2.50 (m, 1 H, 7-H), 3.03 (m, 2 H, 8-H), 3.14 (s, 1 H, propargylCH), 3.97 (AB system, J=18.8 Hz, 2 H, propargyl CH₂), 4.95 (s, 2 H,2-CH₂), 5.79 (t, J=8.6 Hz, 1 H, 6-H), 7.03 (d, J=9.0 Hz, 2 H, 3′-H,5′-H), 7.51 (s, 1 H, 9-H), 7.81 (d, J=6.6 Hz, 2 H, 2′-H, 6′-H), 7.83 (s,1 H, 5-H).

Tri-tert-butylN-{N-{4-[N-((6RS)2-(2,2-dimethylpropionyloxymethyl)-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)-amino]benzoyl}-L-γ-glutamyl}-D-glutamate

A solution of4-[N-((6RS)-2-(2,2-dimethylpropionyloxymethyl)-4oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoicacid (80 mg, 0.15 mmol) in anhydrous dimethylformamide (7 ml) wastreated with tri-tert butyl-L-γ-glutamyl-D-glutamate (150 mg, 0.33mmol), diethyl cyanophosphonate (0.06 ml, 0.40 mmol) and triethylamine(0.06 ml, 0.40 mmol). The solution was stirred at room temperature underargon with protection from the light for 2.5 h. The solution waspartitioned between ethyl acetate (25 ml) and water (25 ml). The aqueouslayer was extracted with ethyl acetate (2×20 ml). The combined organicextracts were washed with 10% aqueous citric acid (2×30 ml), saturatedaqueous NaHCO₃ (30 ml), dilute brine (30 ml), dried (Na₂SO₄) and thesolvent removed in vacuo. The residue was purified by columnchromatography (30 g of silica gel) eluting with 40% ethyl acetate inCH₂Cl₂ to yield the desired product as a white solid (94 mg, 62%); m.p.109° C.; ¹H-NMR (CDCl₃) δ 1.29 (s, 9 H, —COCMe₃), 1.43 (s, 9 H,COOCMe₃), 1.47 (s, 9 H, COOCMe₃), 1.48 (s, 9 H, COOCMe₃), 1.60-2.10 (m,5 H, 2×glu β-CH₂, 7-CH), 2.21 (s, 1 H, propargyl CH), 2.22-2.50 (m, 4 H,2×glu γ-CH₂), 2.59 (m, 1 H, 7-H), 3.08 (n, 1 H, 8-H), 3.20 (m, 1 H,8-H), 3.92 (AB system, J=19.0 Hz, 2 H, propargyl CH₂), 4.48, 4.76 (2×m,2 H, 2×glu α-CH), 5.12 (s, 2 H, 2-CH₂), 5.64 (t, J=8.1 Hz, 1 H, 6-H),6.99 (d, J=8.8 Hz, 2 H, 3′-H, 5′-H), 7.07 (m, 2 H, 2×CONH), 7.64 (s, 1H, 9-H), 7.80 (d, J=8.8 Hz, 2 H, 2′-H, 6′-H), 8.13 (s, 1 H, 5-H); MS(ESI, m/z) 922 {(M+Na)⁺, 100%}, 900 {(M+H)⁺, 40%}; Found C, 64.85; H,7.23; N, 7.33. C₄₉H₆₅N₅O₁₁0.5H₂O requires C, 64.76;H, 7.27, N, 7.71%.

N-{N-{4[N-((6RS-2-Hydroxymethyl4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}-D-glutamicAcid

Tri-tert-butylN-{N-{4-[N-((6RS)-2-(2.2-dimethylpropionyloxymethyl)-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]-benzoyl}-L-γ-glutamyl}-D-glutamate(80 mg, 0.09 mmol) was dissolved in trifluoroacetic acid (5 ml) andstirred at room temperature with protection from the light for 1 h. Thesolvent was removed in vacuo and the residue dissolved in methanol (3ml) and water (3 ml). The pH of the solution was adjusted to pH 12 with1M sodium hydroxide solution and stirred at room temperature for 6 h.The solution was acidified to pH 4 with 1 M hydrochloric acid and cooledto 0° C. The precipitate was collected by filtration and dried undervacuum over P₂O₅ to yield the desired product as a pale brown solid (27mg, 47%); m.p. 172° C.; ¹H-NMR (DMSO-d₆) δ 1.60-2.10 (m, 5 H, 2×gluβ-CH₂, 7-CH), 2.15-2.40 (m, 5H, 2×glu γ-CH₂, 7-H), 2.99 (m, 1 H, 8-H),3.12 (s, 1 H, propargyl CH), 3.16 (m, 1 H, 8-H), 3.98 (AB system, J=19.9Hz, 2 H, propargyl CH₂), 4.18, 4.30 (2×m, 2 H, 2×glu α-CH), 4.36 (s, 2H, 2-CH₂), 5.58 (br s, 1 H, —OH), 5.77 (t, J=7.9 Hz, 1 H, 6-H), 7.01 (d,J=8.9 Hz, 2 H, 3′-H, 5′-H), 7.54 (s, 1 H, 9-H), 7.80 (d, J=8.5 Hz, 2 H,2′-H, 6′-M), 7.82 (s, 1 H, 5-H), 8.15 (d, J=7.5 Hz, 1 H), 8.35 (d, J=7.2Hz, 1 H) (2×CONH); MS (ESI, m/z) 670 {(M+Na)⁺, 45%}, 648 {(M+H)⁺, 100%};HRMS: measured 648.2313; calculated for C₃₂H₃₅N₅O₁₀ (M+H)⁺; 648.2306.

EXAMPLE 2 Synthesis of CB300947

Tert-Butyl 4-[N(5-acetamido-6-bromoindan-1-yl)amino]-2-fluorobenzoate

To a solution of 5-acetamido-6-bromoindan-1-one (0.370 g, 1.38 mmol) inanhydrous methanol (32 ml) was added tert-butyl 4-amino-2-fluorobenzoate(V. Bavetsias et al, J Med. Chem. 1996, 39, 73-85; 0.322 g, 1.52 mmol)followed by decaborane (0.030 g). The reaction mixture was stirred atroom temperature for 11 hours then more decaborane (0.005 g) was addedand stirring was continued for a longer 12 hours under argon. Thesolvent was removed in vacuo, and the residue was purified by columnchromatography eluting with a gradient of ethyl acetate in hexane (30 to40%). The desired compound was obtained as a white solid 0.455 g (71%)m.p.>70° C. (softens); ¹H-NMR (250 MHz, CDCl₃, TMS) 1.57 (s, 9 H,C(CH₃)₃), 2.24 (s, 3H, COCH₃), 1.98, 2.56 (2×m, 2 H, indanyl 2-H), 2.94(m, 2 H, indanyl 3-H), 4.33(d, J=7.90 Hz, 1 H, N-H), 4.99 (q, J=7.06 Hz,1 H, indanyl 1-H), 6.37 (m, 2 H, 3,5-H), 7.48, 8.25, 7.60 (3×s, each 1H, indanyl 4 H, 7-H, CONH), 7.72 (t, J=8.75 Hz, 6-H); MS (ESI, m/z) 485,487 {(M+Na)⁺, bromine isotopic pattern}.

Tert-butyl 4-[N-(5-acetamido-6-cyanoindan-1-yl)amino]-2-fluorobenzoate

To a solution of tert-butyl4-[N-(5-acetamido-6-bromoindan-1-yl)amino]-2-fluorobenzoate (0.420 g,0.90 mmol) in NMP (10 ml) was added copper(l) cyanide (0.137 g, 1.53mmol). The reaction mixture was placed in an oil-bath preheated to 145°C. and stirred at this temperature for 2 hours. The reaction mixture wasallowed to cool to room temperature, then poured into a mixture ofaqueous ammonia (d=0.88, 5 ml) and ice (˜15 ml) and the resulting brownmixture was stirred at room temperature for ˜5 min. The brown solid wascollected by filtration washed with water, then suspended indichloromethane (60 ml). The mixture was stirred at room temperature for5 min, dried (Na₂SO₄), and concentrated in vacuo. Purification by columnchromatography, on elution with 40% ethyl acetate in hexane, afforded asolid that was triturated with diethyl ether/hexane. The desiredcompound was obtained as a white solid: 0.202 g, (55%) m.p. 172-173° C.;¹H-NMR (250 MHz, CDCl₃, TMS) 1.57 (s(obscured by water peak), 9H,C(CH₃)₃), 2.27 (s, 3H, COCH₃), 1.96, 2.65 (2×m, 2 H, indanyl 2-H), 3.00(m, 2 H, indanyl 3-H), 4.30 (d, J=8.40 Hz, 1 H, N-H), 4.99 (q, J=7.80Hz, 1 H, indanyl 1-H), 6.38 (m, 2 H, 3,5-H), 7.52, 8.33, 7.62 (3×s, each1 H, indanyl 4-H, 7-H, CONH), 7.74 (t, J=8.60 Hz, 1 H, 6-H); MS (ESL,m/z) 432 {(M+Na)⁺, 100%}; Found: C, 67.44; H, 5.88; N, 10.25; P, 4.63;C₂₃H₂₄FN₃O₃ requires C, 67.47; H, 5.91; N, 10.26%; F, 4.64%.

Tert-Butyl4-{N-[(6RS)-2-methyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta-[g]quinazolin-6yl]amino}-2-fluorobenzoate

A mixture of tert-butyl4-[N-(5-acetamido-6-cyanoindan-1-yl)amino]-²-fluorobenzoate (0.182 g,0.44 mmol), ethanol (2 ml), and water (0.4 ml) was cooled in anice-bath, then 30% aqueous H₂O₂ solution (0.37 ml) was added followed bygranulated sodium hydroxide pellets (0.030 g, 0.75 mmol). The reactionmixture was stirred at ˜0° C. for 10 min, then it was placed in an oilbath preheated to 55° C. and stirred at this temperature for 30 min Thereaction mixture was allowed to cool to room temperature, then thesolvents were removed in vacuo and the residue was suspended in water(˜15 ml). The pH of this mixture was adjusted to ˜4 with 1N hydrochloricacid. The white precipitate was collected by filtration, washed withwater, and dried in vacuo over P₂O₅. The desired compound was obtainedas a white solid 0.155 g (85%), m.p. 150-152° C.; ¹H-N (250 MHz,DMSO-d₆, TMS) 1.50 (s, 9 H, C(C₃)₃), 2.32 (s, 3 H, 2-CH₃), 1.83, 2.53(2×m, 2 H, 7-H), 3.00 (m, 2 H, 8-H), 5.15 (q, J=7.40 Hz, 1 H, 6-H), 6.57(m, 2 H, 3′,5′-H), 7.16 (d, J=7.75 Hz, 1 H, N¹⁰—H), 7.44, 7.87 (2×s,each 1 H, 5-H, 9-H), 7.59 (t, J=8.73 Hz, 6′-H), 12.11 (s, 1 H, N³-H), MS(ESI, m/z) 819 {(2M+H)⁺, 100%}, 432 {(M+Na)⁺, 10%}, 410 {M+H)⁺, 15%};Found: C, 66.81; H, 5.89, N, 10.11; F, 4.58; C₂₃H₂₄FN₃O₃0.25H₂O requiresC, 66.74; H, 5.96; N, 10.15%; F, 4.59%.

(Propargyl)Co₂(CO)₆ ⁺BF₄ ⁻

This was prepared as in Example 1 from the dicobalthexacarbonylpropargyl alcohol complex. It was used immediately in the next reactionwithout any further purification.

Tert-Butyl4-{N-[(6RS)-2-methyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta-[g]quinazolin-6-yl]-N-(prop-2-ynyl)amino}-2-fluorobenzoate

To a round-bottomed flask containing (propargyl)Co₂(CO)₆ ⁺BF₄ ⁻(0.174 g,0.43 mmol) was added anhydrous dichloromethane (dried by distillationover P₂O₅; 14 ml). The solution was stirred at room temperature for fewminutes under argon, then tert-butyl4-{N-[(6RS)-2-methyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]-quinazolin-6-yl]amino}-2-fluorobenzoate(0.135 g, 0.33 mmol) was added. Stirring was continued at thistemperature for 5 min then diisopropylethylamine (0.4 ml) was added andthe reaction mixture was stirred at room temperature for 25 min underargon. The reaction mixture was partitioned between ethyl acetate (80ml) and brine (40 ml). The organic layer was washed with 10% aqueouscitric acid (30 ml), brine (40 ml), dried (Na₂SO₄), and concentrated invacuo. Purification by column chromatography, on gradient elution withethyl acetate in dichloromethane (25 to 50%), gave a red solid 0.140 g(58%); ¹H-NMR (250 MHz, CDCl₃, TMS) 1.58 (s, 9 H, C(CH₃)₃), 2.54 (s, 3H,2-Mc), 2.32, 2.61 (m, each 1 H, 7-H), 3.03, 3.23 (m, each 1 H, 8-H),4.55 (ABq, J=16.92 Hz, 2 H, N¹⁰—CH₂), 5.59 (t, J=8.30, 1 H, 6-H), 6.00(s, 1 H, propargyl complex C—H), 6.59 (d, J=14.52 Hz, 1 H, 3′-H), 6.69 (d, J=8.70 Hz, 1 H, 5′-H, 7.58, 7.98 (s, each 1 H, 5-H, 9-H), 7.79 (d,J=8.82 Hz, 6′-H), 10.96 (s, 1 H, N³-H). To a solution of this complex(0.100 g, 0.136 mmol) in ethanol (15 ml) was added Fe(NO₃)₃.9H₂O (˜2 g).The clear solution was stirred at room temperature for 5 min then asecond portion of Fe(NO₃)₃.9H₂O (˜1.0 g) was added. The reaction mixturewas stirred at room temperature for a longer 25 min then a final portionof Fe(NO₃)₃.9H₂O (˜1.2 g) was added. Stirring was continued at roomtemperature for an extra 35 min, then the reaction mixture waspartitioned between ethyl acetate (70 ml) and water (30 ml). The organiclayer was washed with brine (2×30 ml), dried (Na₂SO₄), and concentratedin vacuo to leave a crispy solid. Purification by column chromatography,on elution with 5% methanol in chloroform, afforded a white solid; 0.040g (67%), m.p 248-250 ° C., ¹H-NMR (250 MHz, DMSO-d₆, TMS) 1.50 (s, 9H,C(CH₃)₃), 2.32 (s, 3H, 2-CH3), 2.15 (m), 2.50 (m(obscured) (2 H, 7-H),2.90-3.20 (m, 3 H, 8-H, C≡CH), 3.95 (ABq, J=18.52 Hz, 2 H, CH₂C≡C), 5.74(t, J=7.70 Hz, 1 H, 6-H), 6.80 (m, 2 H, 3′,5′-H), 7.48, 7.76 (2×s, each1 H, 5-H, 9-H), 7.68 (t, J=9.01 Hz, 6′-H), 12.10 (s, 1 H, N³-H); MS(ESI, m/z) 470 {(M+Na)⁺, 55%}, 448 {M+H)⁺, 70%}; FAB-HRMS; measured:470.1840, calculated for C₂₆H₂₆FN₃O₃Na: 470.1856.

N-[(6RS)-2-Methyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl]-N-(prop-2-ynyl)amino)-2-fluorobenzoicacid

A solution of tert-butyl4-{N-[(6RS)-2-methyl4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl]-N-(prop-2-ynyl)amino}-2-fluorobenzoate(0.061 g, 0.14 mmol) in dichloromethane (1 ml) and trifluoroacetic acid(2.4 ml) was stirred at room temperature for 1.5 hours, then thesolvents were removed in vacuo. The residue was triturated with diethylether and the precipitate was collected by filtration, washed withdiethyl ether and dried in vacuo over P₂O₅ to afford the desiredcompound as the trifluoroacetate salt (0.046 g). ¹H-NMR (250 MHz,DMSO-d₆, TMS) 2.33 (s, 3H, 2-CH₃), 2.18 (m), 2.50 (m(obscured)) (2 H,7-H), 2.85-3.20 (m, 3H, 8-H, C≡CH), 3.95 (ABq, J=19.02 Hz, 2 H, CH₂C≡C),5.76 (t, J=7.95 Hz, 1 H, 6-H), 6.80 (d, J=16.40 Hz, 1 H, 3′-H), 6.84 (d,J=9.46 Hz, 1 H, 5′-H), 7.48, 7.78 (2×s, each 1 H, 5-H, 9-H) 7.74 (t,J=9.02 Hz, 6′-H). 12.14 (s, 1 H, N³-H); MS ESI, m/z) 783 {(2M+H)⁺,100%}, 392 {(M+H)⁺, 55%}.

Tri-tert-butylN-{N-{4[N-((6RS)-2-methoxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6yl)N-(prop-2-ynyl)amino]-2-fluorobenzoyl}-L-γ-glutamyl}-D-glutamate

To a solution of tri-tert-butyl L-γ-glutamyl-D-glutamate (V. Bavetsiaset al, J Med. Chem. 1996, 39, 73-85; 0.066 g, 0.14 mmol) in anhydrousDMF (2.5 ml) was added.

4-{N-[(6RS)-2-methyl4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl]-N-(prop-2-ynyl)amino}-2-fluorobenzoicacid trifluoroacetate salt (0.045 g, 0.11 mmol), followed by diethylcyanophosphonate (0.051 g, 0.31 mmol) and triethylamine (0.032 g, 0.32mmol). The reaction mixture was stirred at room temperature for 2.5hours, then it was partitioned between ethyl acetate (150 ml) and water(100 ml). The aqueous layer was extracted with more ethyl acetate (100ml). The combined organics were washed with 10% aqueous citric acid(2×50 ml), saturated sodium bicarbonate solution (2×50 ml), and brine(50 ml), then dried (Na₂SO₄), and concentrated in vacuo. Purification bycolumn chromatography, on elution with 1% methanol in ethyl acetate,afforded a white solid: 0.077 g (67 %); m.p.>110° C. (softens); ¹H-NMR(250 MHz, DMSO-d₆, TMS) 1.37, 1.38, 1.41 (3×s, 27 H, 3×C(CH₃)₃),1.60-2.35 (m, 9 H, 2×β-CH₂, 2×γ-CH₂, 7-H), 2.32 (s, 3 H, 2-CH₃), 2.52 (mobscured by DMSO peak, 1 H, 7-H), 2.86-3.23 (m, 3 H, C≡CH, 8-H), 3.97(ABq, J=19.0 Hz, 2 H, CH₂C≡C), 4.12, 4.30 (2×m, 2 H, 2×α-CH), 5.74 (t,J=8.70 Hz, 1 H, 6-H), 6.80 (d, J=14.20 Hz, 1 H, 3′-H), 6.85 (d, J=8.07Hz, 1 H, 5′-H), 7.48 (s, 1 H, 9-H), 7.58 (t, J=8.8 Hz, 1 H, 6′-H), 7.78(s, 1 H, 5-H), 7.98 (t, J=6.42 Hz, 1 H, CONH), 8.13 (d, J=7.4 Hz, 1 H,CH₂CONH), 12.11 (s, 1 H, N³-H); (ESI, m/z) 818 {(M+H)⁺, 100%}; Found C,64.34; H, 7.09; N, 8.20; F, 2.22, C₄₄H₅₆FN₅O₉ requires C, 64.61; H,6.90; N, 8.56; F, 2.32%.

N-{N-{4-[N-((6RS)-2methyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]-quinazolin-6-yl)-N-(prop-2-ynyl)amino]-2-fluorobenzoyl)-L-γ-glutamyl}-D-glutamicacid

A solution of tri-tert-butylN-{N-{4-[N-((6RS)-2-methyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]-2-fluorobenzoyl}-L-γ-glutamyl}-D-glutamate(0.066 g, 0.08 mmol) in trifluoroacetic acid (4.5 ml) was stirred atroom temperature for 1 hour and 10 min with protection from the light.The solvent was then removed in vacuo and the residue was suspended inwater (5 ml). The pH was adjusted to ˜12 with 1N NaOH, then to ˜4 with1N hydrochloric acid. The white precipitate was collected by filtrationand dried in vacuo over P₂O₅: 0.032 g (63%), m.p. 175° C. (dec); ¹H-NMR(250 MHz, DMSO-d₆, TMS) 1.65-2.25 (m, 9 H, 2×β-CH₂, 2×γ-CH₂, 7-H), 2.32(s, 3H, 2-CH₃), 2.52 (m obscured by DMSO peak, 1 H, 7-H), 2.90-3.22 (m,3H, C≡CH, 8-H), 3.95 (ABq, J=19.15 Hz, 2 H, CH₂C≡C), 4.18, 4.37 (2×m,2H, 2×α-CH), 5.74 (t, J=8.30 Hz, 1 H, 6-H), 6.81 (d, J=15.50 Hz, 1 H,3′-H), 6.85 (d, J=7.88 Hz, 1 H, 5′-H), 7.48 (s, 1 H, 9-H), 7.62 (t,J=8.8 Hz, 1 H, 6′-H), 7.78 (s, 1 H, 5-H), 7.97 (t, J=6.55 Hz, 1 H,CONH), 8.12 (d, J=8.05 Hz, 1 H, CH₂CONH), 12.11 (s, 1 H, N³-H); (ESI,m/z) 650 {(M+H)⁺, 100%}; FAB-HRMS, measured: 672.2060; calculated forC₃₂H₃₂FN₅O₉Na: 672.2082.

EXAMPLE 3 Synthesis of CB300960 (N-methyl Derivative of CB300945)

4-{N-[(6RS)-2-Hydroxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6yl]-N-(prop2-ynyl)amino}BenzoicAcid

Method A: A solution of tert-butyl 4-{N-[(6RS)-2-(2,2-dimethylpropionyloxymethyl)-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl]-N-(prop-2-ynyl)amino}benzoate(0.150 g, 0.28 mmol) in dichloromethane (2 ml) and trifluoroacetic acid(6 ml) was stirred at room temperature for 1 hour. The solvents werethen removed in vacuo, and the residue was suspended in methanol (3 ml)and water (5 ml). The pH was adjusted to ˜10 with 1 N NaOH (1.1 ml), andthe mixture was stirred at room temperature for 4 hours. The reactionmixture was diluted with water (5 ml) and the pH was adjusted to ˜5 with1N HCI. The solid was then collected by filtration, but ¹H-NMR indicatedno complete removal of the pivaloyl group. This solid was suspended intothe filtrate and then 1N NaOH (0.9 ml, 0.9 mmol) was added (pH˜12). Themixture was stirred at room temperature for 3.5 hours, then more 1N NaOH(0.2 ml) was added, and the mixture was stirred at room temperature fora further 0.5 hours. The pH was then adjusted to ˜5.0 with 1N HCl. Theoff-white precipitate was collected by filtration, washed with water,and dried in vacuo over P₂O₅: 0.086 g, (79%); ¹H-NMR (250 MHz, DMSO-d₆,TMS) 2.22 (m, 1 H 7-CH), 2.90-3.30 (m, 3 H, C≡CH, 8-H), 3.97 (ABq,J=18.6 Hz, 2 H, CH₂C≡C), 4.37 (d, J=6.1 Hz, 2 H, 2-CH₂), 5.56 (t, 1 H,CH₂OH), 5.78 (t, J=7.51 Hz, 1 H, 6-H), 7.03 (d, J=8.9 Hz, 2 H, 3′,5′-H),7.55 (s, 1 H, 9-H), 7.82 (m, 3 H, 2′,6′-H, 5-H), MS (ESI, m/z) 779{(2M+H)⁺, 100%}, 390 {(M+H)⁺, 60%}.

Method B: A solution of tert-butyl4-{N-[(6RS)-2-hydroxymethyl-4oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl]-N-(prop-2-ynyl)amino}-benzoate(0.050 g, 0.11 mmol) in dichloromethane (1 ml) and trifluoroacetic acid(2.4 ml) was stirred at room temperature for 1 hour. The solvents werethen removed in vacuo, and the residue was triturated with diethylether. The off-white precipitate was collected by filtration, and washedwith other to obtain the desired product as the trifluoroacetate salt:0.044 g.

Tri-tert-butylN-{N-{4-[N-((6RS)-2-hydroxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}-N-methyl-L-glutamate

To a mixture of4-{N-[(6RS)-2-hydroxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl]-N-(prop-2-ynyl)amino}benzoicacid (0.075 g, ˜0.19 mmol), tri-tert-butylL-γ-glutamyl-N-methyl-L-glutamate (V. Bavetsias et al., J Med. Chem.,1997, 40, 1495-1510; 0.110 g, 0.24 mmol), and anhydrous DMF (2.0 ml) wasadded diethyl cyanophosphonate (0.036 g, 0.22 mmol) with the aid ofanhydrous DMF (0.2 ml) followed by triethylamine (0.022 g, 0.22 mmol).The clear solution was stirred at room temperature for 1.5 hours, thenit was partitioned between ethyl acetate (50 ml) and brine (40 ml). Theaqueous layer was extracted with more ethyl acetate (2×50 ml). Thecombined organics were washed with 10% aqueous citric acid (40 ml),saturated sodium bicarbonate solution (40 ml), and brine (40 ml), dried(Na₂SO₄), and concentrated in vacuo. Purification by columnchromatography, on elution with a gradient of methanol indichloromethane (0 to 6%), afforded an off-white solid that was furtherpurified by trituration with hexane/dichloromethane/diethyl ether: 0.062g (40%); mp 116-120° C. (softens); ¹H-NM (250 MHz, DMSO-d₆, TMS) 1.36,1.37, 1.38, 1.41 (4×s, 27H, 3×C(CH₃)₃), 1.70-2.35 (m) and 2.50 (mobscured by DMSO peak) (10 H, 2×γ-CH₂, 2×γ-CH₂, 7-CH₂), 2.63, 2.82 (2×s,3H, CONMe), 2.90-3.25 (m, 3H, C≡CH, 8-H), 3.97 (ABq, J=17.6 Hz, 2 H,CH₂C≡C), 4.32 (m, 1 H, glu α-CH), 4.38 (d, J=6.1 Hz, 2 H, 2-CH₂), 4.50,4.82 (2×dd, 1 H, Meglu α-CH), 5.56 (t, J=6.9 Hz, 1 H, CH₂OH), 5.78 (t,J=7.10 Hz, 1 H, 6-H), 7.02 (d, J=8.6 Hz, 2 H, 3′,5′-H), 7.55 (s, 1 H,9-H), 7.78 (d, J=8.9 Hz, 2 H, 2′,6′-H), 7.82 (s, 1 H, 5-H), 8.32 (m, 1H, CONH), 11.81 (s, 1 H, N³-H); MS (ESI, m/z) 830((M+H)⁺, 100%}.

N-{N-{4-[N-((6RS)-2-hydroxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}-N-methyl-L-glutamicacid

A solution of tri-tert-butylN-{N-{4-[N-((6RS)-2-hydroxymethyl-4-oxo-3,4,7,8-terahydro-6H-cyclopenta[g]quinazolin-6yl)-N-(prop-2-ynyl)amino]-benzoyl}-L-γ-glutamyl}-N-methyl-L-glutamate(0.060 g, 0.07 mmol) in trifluoroacetic acid (3.5 ml) was stirred atroom temperature for 1 hour and 10 min with protection from the light.The solvent was then removed in vacuo and the residue was suspended inwater (6 ml). The pH was adjusted to ˜10 with 1N NaOH, then to ˜4 with1N hydrochloric acid. The white precipitate was collected by filtration,and dried in vacuo over P₂O₅: 0.035 g (77%), mp>165° C. (dec); ¹H-NMR(250 MHz, DMSO-d₆, TMS) 1.80-2.35 (m) and 2.50 (m obscured by DMSO peak)(10 H, 2×β-CH₂, 2×γ-CH₂, 7-CH₂), 2.66, 2.83 (2×s, 3H, CONMe), 2.90-3.25(m, 3H, C≡CH, 8-H), 3.97 (ABq, J=18.4 Hz, 2 H, CH₂C≡C), 4.32 (mobscured, 1 H, glu α-CH), 4.38 (d, J=5.6 Hz, 2 H, 2-CH₂), 4.55, 4.91(2×dd, J=10.0, 4.5 Hz, 1 H, Meglu α-CH), 5.56 (poorly resolved t, 1 H,CH2OH), 5.77 (t, J=8.06 Hz, 1 H, 6-H), 7.02 (d, J=7.8 Hz, 2 H, 3′,5-H),7.55 (s, 1 H, 9-H), 7.81 (d, J=10.1 Hz, 3H, 2′,6′-H, 5-H), 8.32 (m, 1 H,CONH), 11.82 (s, 1 H, N³-H); MS (ESI, m/z) 662 {(M+H)⁺, 100%}; Found: C,57.52; H, 5.52; N, 10.17; C₃₃H₃₅N₅O₁₀1.5 H₂O requires: C, 57.55; H,5.56; N, 10.17%.

EXAMPLE 4 In Vitro Evaluation

The cyclopenta[g]quinazolines of the invention, particularly those withL-glu-γ-D-glu ligands, or modified ligands (e.g. CB300907 and CB300899)are potent inhibitors of TS (Kiapp 0.17 to 3 nM) and have very lowaffinities for the reduced-folate carrier (RFC) (Table 1). Theaffinities relative to folic acid (Table 1) varied little and wereslightly lower than folic acid itself These properties did notnecessarily predict for in vitro potency and selectivity for human celllines overexpressing the α-FR. For example, CB300944, a 2-NH₂ analogue,has low potency and no selectivity for either A431-FBP or KB cells. Thebest compounds in this regard are the 2-CH₃ and 2-CH₂OH compounds. Thesewere 150 to 4700-fold more active in A431-FBP cells compared with theA431 cells Similarly, when an excess of folic acid was added to blockFR-mediated uptake in the A431-FBP cells, the difference ± folic acidwas 100 to 4300-fold. In KB cells the difference ± folic acid was 73 to2,100. It should be noted that all these experiments were performed inmedia containing 20 nM 5-formyl tetrahydrofolate (leucovorin; LV) as thefolate source. This is in the physiological range of folate in humanplasma. If experiments are done in low folate, 1 nM LV or lower (as manyantifolate experiments in the literature are) then the degree ofselectivity increases for several of the quinazoline-based compoundsincluding CB3717.

Tables 4, 5, 6 and 7 show the structures of the compounds tested.

Example 5 Formulation

The following illustrate representative pharmaceutical dosage formscontaining a cyclopenta[g]quinazoline of formula (I), particularly inpharmaceutically acceptable salt form, for therapeutic or prophylacticuse in humans: (a) Tablet I mg/tablet Cyclopenta[g]quinazoline salt 100Lactose Ph.Eur. 182.75 Croscarmellose sodium 12.0 Maize starch paste (5%w/v paste) 2.25 Magnesium stearate 3.0 (b) Tablet II mg/tabletCyclopenta[g]quinazoline salt 50 Lactose Ph.Eur. 223.75 Croscarmellosesodium 6.0 Maize starch 15.0 Polyvinylpyrrolidone (5% w/v paste) 2.25Magnesium stearate 3.0 (c) Tablet III mg/tablet Cyclopenta[g]quinazolinesalt 1.0 Lactose Ph.Eur. 93.25 Croscarmellose sodium 4.0 Maize starchpaste (5% w/v paste) 0.75 Magnesium stearate 1.0 (d) Capsule mg/capsuleCyclopenta[g]quinazoline salt 10.0 Lactose Ph.Eur. 488.5 Magnesiumstearate 1.5 (e) Injection I (50 mg/ml) Cyclopenta[g]quinazoline salt5.0% w/v 1 M Sodium hydroxide solution 15.0% v/v  0.1 M Hydrochloricacid (to adjust pH to 7.6) Polyethylene glycol 400 4.5% w/v Water forinjection to 100% (f) Injection II (10 mg/ml) Cyclopenta[g]quinazolinesalt 1.0% w/v Sodium phosphate BP 3.6% w/v 0.1 M Sodium hydroxidesolution 15.0% v/v  Water for injection to 100% (g) Injection III (1mg/ml. buffered to pH 6) Cyclopenta[g]quinazoline salt 0.1% w/v Sodiumphosphate BP 2.26% w/v  Citric acid 0.38% w/v  Polyethylene glycol 4003.5% w/v Water for injection to 100%

The above formulations may be prepared by conventional procedures wellknown in the pharmaceutical art. The tablets (a) to (c) may be entericcoated by conventional means, for example with a coating of celluloseacetate phthalate. TABLE 1 In vitro properties of quinazolines andcyclopenta[g]quinazolines Inhibition of ¹Inhibition of ³H MTX ²Inverserelative isolated L1210 uptake Ki (μM) affinity for α-FR Ligand TS,Kiapp (nM) ³L1210 ⁴W1L2 ⁵L1210-FBP ⁶A431-FBP Comparative compounds*Raltitrexed 2-CH₃ L-Glu 510 2.6, 2.3 0.61, 0.70 0.6 ± 0.1 0.24 ± 0.02*CB3717 2-NH₂ L-Glu 20 46 ± 17 20 ± 16  1.5 ± 0.12  1.2 ± 0.06*IC1198583 2-CH₃ L-Glu 31  2.6 ± 0.06 1.2, 1.0 0.80 ± 0.05 CB3900 2-CH₃,7-CH₃ L-Glu 23 4.3, 4.7 1.3 0.30 ± 0.07 CB300464 2-CH₃, L-Glu 3 9.1 ±5.1 4.9 ± 2.3 0.44 ± 0.10  0.51 ± 0.0058 (S) cyclopentane CB3009582-NH₂, L-glu 0.75  0.73 ± 0.035  0.57 ± 0.059 (R, S) cyclopentaneCB300959 2-CH₂OH, L-glu 12  0.71 ± 0.012  0.57 ± 0.032 (R, S)cyclopentane ZD9331 2-CH₃, L-Glu-γ-tet 1.0 2.4 ± 1.0 0.76 ± 0.11 0.54 ±0.1  0.30 ± 0.08 7-CH₃, 2′F CB30901 2-CH₃, 7-CH₃ L-glu-γ-D-glu 2.0 269 ±162 83  0.31 ± 0.0089 — CB300944 2-NH₂, L-glu-γ-D-glu0.67 >250 >250, >250 0.73 ± 0.05 0.68 ± 0.10 (R, S) cyclopentaneCompounds of the invention CB300638 2-CH₃, L-glu-γ-D-glu 0.33 ±0.16 >250 115 ± 12  0.66 ± 0.08 0.53 ± 0.07 (S) cyclopentane CB3006382-CH₃, L-glu-γ-D-glu 0.42 166 ± 34  279 ± 146 0.60 ± 0.06 0.59 ± 0.03(R, S) cyclopentane CB300947 2-CH₃, 2′F, L-glu-γ-D-glu 0.46, 0.77 >25097,315 0.73 ± 0.01  0.58 ± 0.017 (R, S) cyclopentane CB300945 2-CH₂OH,L-glu-γ-D-glu 1.9 — —  0.70 ± 0.015 0.69 ± 0.05 (S) cyclopentaneCB300945 2-CH₂OH, L-glu-γ-D-glu 2.6, 3.3 >250 >250 0.73 ± 0.06 0.67 ±0.11 (R, S) cyclopentane CB300907 2-CH₃, L-glu-γ-D-glu(α- 0.16, 0.17 186± 121 142 ± 54  0.61 ± 0.07  0.58 ± 0.032 (S) cyclopentane tetrazole)CB300899 2-CH₃, L-glu-NCH₃-L- 0.78 >250 >250 0.62 ± 0.04  0.64 ± 0.046(R, S) cyclopentane glu*can be polyglutamated¹High Ki = low affinity for the reduced-folate carrier (RFC);²High Inverse relative affinity = high affinity for the α-FR. Folic acid= 1;³mouse L1210 tumor cells;⁴human lymphoblastoid cells;⁵L1210-FBP mouse tumor cells that overexpress the α-FR (Jansen et al.,Cancer Res., 49, 2455-2459, 1989-later identified as L1210 origin: seecorrection in Cancer Res. 55, 1995);⁶A431-FBP cells are human A431 tumor cells transfected with the α-FR(Bagnoli et al., Oncogene 19, 4754-4763, 2000)

TABLE 2 Activity of quinazolines and cyclopenta[g]quinazolines in humanA431 and A431-FBP cell lines grown in 20 nM folate (R, S LV) Inhibitionof cell growth, IC₅₀, uM Inhibition of cell growth, IC₅₀, uM 20 nM LV 20nM LV ¹A431 + 1 μM FA ²A431-FBP ¹A431-FBP + 1 μM FA (fold increased IC₅₀in (fold increased sensitivity (fold increased IC₅₀ in A431 presence offolic acid) compared with A431) presence of folic acid) Comparativecompounds *Raltitrexed 0.0034 ± 0.0016 0.0036 ± 0.0016 0.00079 ± 0.00036 0.0013 ± 0.00070 (1) (4) (2) *CB3717  1.2 ± 0.63 1.4 ± 0.56 0.26 ± 0.120.45 ± 0.14 (1) (4) (2) *ICI 198583 0.024 ± 0.004  0.026 ± 0.0038 0.012± 0.005  0.020 ± 0.0046 (1) (2) (2) CB3900  2.2 ± 0.26 2.6 ± 0.92  1.4 ±0.36 1.5 ± 0.29 (1) (2) (1) CB300464 (S) 0.76 ± 0.40 0.77 ± 0.41 0.24 ±0.13 0.36 ± 0.17 (1) (3) (2) CB300958 (R, S) 23 23 10 20 (1) (2) (2)CB300959 (R, S) 4.2 5.8 8.8 18 (1) (0.5) (2) ZD9331 0.082 ± 0.042 0.067± 0.029  0.018 ± 0.0097  0.034 ± 0.0087 (1) (4) (2) CB30901  1.4 ± 0.70 1.1 ± 0.76 0.21 ± 0.26 0.47 ± 0.24 (1) (5) (2) CB300944 (R, S) 23, 2722, 25  8.3 ± 0.64 7.1 ± 1.0 (1) (3) (1) Compounds of the inventionCB300638 (S) 0.81 ± 0.31 0.97 ± 0.58 0.0030 ± 0.0021 0.49 ± 0.17 (1)(270) (160) CB300638 (R, S)  1.4 ± 0.23  1.4 ± 0.25 0.0065 ± 0.0001 0.87± 0.29 (1) (220) (130) CB300947 (R, S)  1.4 ± 0.26 1.2 ± 0.51 0.0094 ±0.004 0.93 ± 0.21 (1) (150) (100) CB300945 (S) 7.0 6.5 0.0015 6.4 (1)(4700) (4300) CB300945 (R, S) 9.8 ± 3.4 9.3 ± 3.5 0.0021 ± 0.0011 6.5 ±0.86 (1) (4700) (3100) CB300907 (S)  1.4 ± 0.82 1.6 ± 1.1 0.0026 ±0.0018 0.43 ± 0.18 (1) (540) (170) CB300899 (R, S)  2.2 ± 0.17  2.1 ±0.058 0.00092 ± 0.00012  1.9 ± 0.17 (1) (2400) (2100) CB300960 (R, S)4.5 4.3 0.028 5.1 (1) (180)*can be polyglutamated; FA = folic acid¹Folic acid is added in excess to compete with compounds for binding tothe α-FR. FIGS. >1 in parentheses indicate α-FR-mediated uptake andgrowth inhibition;²A431-FBP cells are transfected with the α-FR, and the figures inparentheses >1 indicate increased sensitivity relative to A431 cells andα-FR-mediated uptake. Higher numbers represent higher selectivity.

TABLE 3 Activity of quinazolines and oyclopenta[g]quinazolines in humanKB cells grown in 20 nM folate (R, S LV) Inhibition of cell growth,IC₅₀, μM 20 nM LV ²KB + 1 μM FA (fold increased IC₅₀ in ¹KB presence offolic acid) Comparative compounds Raltitrexed  0.0011 ± 0.00071  0.0012± 0.00059 (1) CB3717 0.007 ± 0.002 0.58 ± 0.20 (80) ICI 198583  0.002 ±0.0004  0.02 ± 0.002 (10) CB3900  0.13 ± 0.006 0.31 ± 0.04 (2) CB300464(S)  0.009 ± 0.0004 0.21 ± 0.01 (23) CB300958 (R, S) 8.4, 6.8 26, 15 (3)CB300959 (R, S) 0.42, 0.50 5.8, 4.8 (11) ZD9331 0.0036 ± 0.0021 0.01 ±0.005 (3) CB30901  0.010 ± 0.0056  0.21 ± 0.023 (21) CB300944 (R, S)  19 ± 0.58  19 ± 1.2 (1) Compounds of the invention CB300638 (S) 0.0036± 0.0015 0.39 ± 0.18 (110) CB300638 (R, S) 0.0053 ± 0.0025 0.76 ± 0.17(140) CB300947 (R, S) 0.008 ± 0.001 0.58 ± 0.03 (73) CB300945 (S) 0.0027± 0.0015  4.8 ± 0.75 (1800) CB300945 (R, S) 0.0034 ± 0.0009 7.3 ± 2.0(2100) CB300907 (S) 0.0062 ± 0.003  0.49 ± 0.14 (79) CB300899 (R, S) 0.0051 ± 0.00031  2.2 ± 0.40 (430)¹KB cells consitutively overexpress the α-FR²Folic acid is added in excess to compete with compounds for binding tothe α-FR. Figures >1 in parentheses indicate α-FR-mediated uptake andgrowth inhibition. Higher numbers represent a high degree ofα-FR-mediated uptake

TABLE 4 Structures of the compounds tested-Comparative compounds

TABLE 5 Structures of the compounds tested-Comparative compounds

TABLE 6 Structures of the compounds tested-Compounds of the invention

TABLE 7 Structures of the compounds tested-Compounds of the invention

1. Use of a cyclopenta[g]quinazoline of the formula (I):

wherein: A is hydrogen or a group OR⁰ or NR⁰R¹ wherein R⁰ and R¹ areeach independently hydrogen, C₁₋₄ alkyl, C₃₋₄ alkenyl, C₃₋₄ alkynyl,C₂₋₄ hydroxyalkyl, C₂₋₄ halogenoalkyl or C₁₋₄ cyanoalkyl, or R⁰ and R¹together with the intermediate N form a five- or six-memberedheterocyclic ring; p is an integer in the range 1 to 4; R² is hydrogen,C₁₋₄ alkyl, C₃₋₄ alkenyl, C₃₋₄ alkynyl, C₂₋₄ hydroxyalkyl, C₂₋₄halogenoalkyl or C₁₋₄ cyanoalkyl; Ar¹ is phenylene, thiophenediyl,thiazolediyl, pyridinediyl or pyrimidinediyl which may optionally bearone or two substituents selected from halogeno, hydroxy, amino, nitro,cyano, trifluoromethyl, C₁₋₄ alkyl and C₁₋₄ alkoxy; and R³ is a group ofthe formula:—A⁵—CON(R)CH(Y⁴)Y⁵ in which A⁵ is a C₁₋₆ alkylene group and R ishydrogen, C₁₋₄ alkyl, C₃₋₄ alkenyl or C₃₋₄ alkynyl; Y⁴ is carboxy,tetrazol-5-yl, N-(C₁₋₄ alkylsulfonyl)carbamoyl,N-(phenylsulfonyl)-carbamoyl which may optionally bear one or twosubstituents on the phenyl ring selected from the group consisting ofhalogeno, nitro, C₁₋₄ alkyl and C₁₋₄ alkoxy, tetrazol-5-ylthio,tetrazol-5-ylsulfinyl or tetrazol-5-ylsulfonyl; and Y⁵ is the residue ofa naturally occurring amino acid NH₂CH(CO₂ H)Y⁵; or Y⁵ is a group of theformula:—A⁴—CO₂ H in which A⁴ is a C₂₋₆ alkylene group; or Y⁵ is a group of theformula:—A⁶—Ar³—A⁷—Y⁶ in which A⁶ is a bond between the α-carbon atom of thegroup —A⁵—CON(R)CH(Y⁴)— and Ar³ or is a C₁₋₂ alkylene group; Ar³ isphenylene, tetrazolediyl, thiophenediyl, thiazolediyl, pyridinediyl orpyrimidinediyl which in the case of phenylene may optionally bear one ortwo substituents on the ring selected from halogeno, nitro, C₁₋₄ alkyland C₁₋₄ alkoxy; A⁷ is a C₁₋₃ alkylene or C₂₋₃ alkenylene group; and Y⁶is carboxy, tetrazol-5-yl, N-(C₁₋₄ alkylsulfonyl)carbamoyl,N-(phenylsulfonyl)carbamoyl which may optionally bear one or twosubstituents on the phenyl ring selected from the group consisting ofhalogeno, nitro, C₁₋₄ alkyl and C₁₋₄ alkoxy, tetrazol-5-ylthio,tetrazol-5-ylsulfinyl or tetrazol-5-ylsulfonyl; the compound (I)optionally being in the form of a pharmaceutically acceptable salt orester; for the manufacture of a medicament for the treatment of solidtumours.
 2. Use as claimed in claim 1 wherein: R⁰ and R¹ are eachindependently hydrogen or C₁₋₄ alkyl; R² is ethyl, propyl, prop-2-enyl,prop-2-ynyl, 2-hydroxyethyl, 2-fluoroethyl, 2-bromoethyl or2-cyanoethyl; Ar¹ is 1,4-phenylene which may optionally bear one or twosubstituents selected from the group consisting of chloro, fluoro,thiophene-2,5-diyl, thiazole-2,5-diyl or pyridine-2,5-diyl; A⁵ is anethylene group; and Y⁵ is the residue of a naturally occurring aminoacid NH₂CH(CO₂ H)Y⁵.
 3. Use as claimed in claim 2 wherein: A is hydrogenor a group OR⁰ in which R⁰ is hydrogen or methyl; R² is ethyl orprop-2-ynyl; and Ar¹ is 1,4-phenylene or 1,4-phenylene having a2-fluoro.
 4. Use as claimed in claim 3 wherein R³ is the residue of anacid NH₂.CH(COOH)R³ which comprises L-Glu-γ-D-Glu, i.e.N-L-γ-glutamyl-D-glutamic acid, or L-Glu-γ-L-Glu, i.e.N-L-γ-glutamyl-L-glutamic acid wherein R³ is of formula:

R³ is the corresponding N-methyl derivative of formula:

or R³ is the corresponding tetrazol-5-yl derivative of formula:


5. Use as claimed in claim 1 wherein p is
 1. 6. Use as claimed in claim1 wherein the cyclopenta[g]quinazoline of formula (I) comprises:N-{N-{4-[N-((6RS)-2-methyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}-D-glutamicacid;(4R)-4-{N-{N-{4-[N-((6RS)-2-methyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]-quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}amino}-4-(tetrazol-5-yl)-butyricacid;N-{N-{4-[N-((6RS)-2-methoxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta-[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}-D-glutamicacid;N-{N-{4-[N-((6RS)-2-hydroxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta-[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}-D-glutamicacid; orN-{N-{4-[N-((6RS)-2-hydroxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]-quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-▭-glutamyl}-N-methyl-L-glutamicacid; or a pharmaceutically acceptable salt or ester thereof.
 7. Useaccording to claim 1 wherein the cyclopenta[g]quinazoline of formula (I)is administered together with a pharmaceutically acceptable diluent orcarrier.
 8. Use according to claim 1 wherein the medicament is for usein the treatment of a carcinoma of ovarian origin.
 9. A method foraiding regression and palliation of solid tumours in a patient in needof such treatment which comprises administering to said patient aneffective amount of a cyclopenta[g]quinazoline according to claim 1.